【摘要】 目的:探讨炎症微环境对Wistar大鼠骨髓间充质干细胞(rBMSCs)成骨分化Periostin基因表达的影响。方法:采用全骨髓贴壁法分离和培养rBMSCs,取第3代rBMSCs成骨、成脂分化诱导;10μg/L TNF-α作用于rBMSCs模拟炎症微环境,采用CCK-8法检测细胞增殖活性,qRT-PCR检测IL-1β及TNF-α的基因表达。将第3代rBMSCs分成对照组(OM)、炎症组(OM+TNF-α)进行成骨诱导,茜素红染色及钙离子沉积量检测,在成骨诱导的第7、14天采用qRT-PCR检测细胞中Periostin和Runx2的基因表达水平,Western blot检测Periostin蛋白表达量。结果:全骨髓贴壁法成功分离培养rBMSCs;炎症组细胞TNF-α及IL-1β的基因表达水平分别是对照组的1.8倍(P<0.05)、2.1倍(P<0.01);炎症组较对照组成骨能力下降,相关成骨基因表达量降低;成骨诱导后炎症组的Periostin基因及蛋白表达水平较对照组明显降低(P<0.05)。结论:炎症微环境下诱导rBMSCs成骨分化Periostin基因表达水平下降,且rBMSCs的成骨能力降低。更多还原

【Abstract】 Objective: To investigate the expression level of Periostin gene in osteogenic differentiation of wistar rat bone marrow mesenchymal stem cells(rBMSCs) in inflammatory microenvironment. Methods: rBMSCs were isolated and cultured by whole bone marrow adherence method. The cell morphology was observed and the osteogenic differentiation of the third generation rBMSCs was induced. The cell inflammatory response was induced by 10 ng/mL TNF-α. Then, the cell proliferation activity was detected by CCK-8 and the gene expression levels of IL-1β and TNF-α were measured by real-time quantitative PCR. The third generation of rBMSCs was divided into control group and inflammation group for osteogenic induction. The deposition amounts of calcium ions were measured after alizarin red staining. The expression levels of Periostin in the cells were detected by qRT-PCR and Western blot on the 14 th day of osteogenic induction. Results:rBMSCs were successfully isolated and cultured by whole bone marrow adherence method. The gene expression levels of TNF-α and IL-1β in the inflammation group were 1.8 times(P<0.05) and 2.1 times(P<0.01) of those of control group, respectively. Compared with the control group, the inflammation group had lower bone formation ability and lower expression of related osteogenic genes. The expression levels of Periostin gene and protein in the inflammation group after osteoinduction were significantly lower than those in the control group(P<0.05). Conclusion: The expression level of Periostin gene in osteogenic differentiation of rBMSCs induced by inflammatory microenvironment decreased, and the osteogenic capacity of rBMSCs decreased.更多还原